The long-term goals of this work are to understand cellular and molecular mechanisms of endoderm development during vertebrate embryogenesis. Our current understanding of this process has benefited from studies in several model systems including Xenopus, zebrafish and mouse. Our work in zebrafish has identified three transcription factors important for endoderm formation, namely the Mix-type homeodomain factor Bon, the zinc finger factor Gata5 and the Sox-type factor Cas. We further assembled these and other factors into a genetic pathway of endoderm formation. We have recently completed a screen for additional mutations that affect the expression of sox17, an early endodermal marker. This screen has led to the identification of 9 mutants that we are currently recovering. Interestingly, one of these mutations affects the smoothened gene, thereby unexpectedly implicating Hedgehog (Hh) signaling in early endoderm formation. While Hh signaling has previously been implicated in endoderm development, all studies thus far have pointed to a late role for Hh in this process. Our initial results, however, indicate that Hh signaling plays a very early role in endoderm development. We aim to continue our investigation of endoderm development in zebrafish by focusing on this new set of early endoderm mutants and propose the following specific aims: 1) recover and determine the phenotype of the 8 new endoderm mutants; 2) investigate the role of Smoothened/Hh signaling in endoderm formation. Detailed studies of embryos with blocked or enhanced Smoothened/Hh signaling will allow us to study the precise role of this signaling pathway in early endoderm differentiation and patterning; and 3) analyze in detail, including positionally cloning, two genes from our new set of mutants, s421 and s468, that regulate endoderm formation in a previously undocumented manner. These molecular genetic studies of endoderm formation will help provide the necessary context for further investigating the pathophysiology of malformations and malfunctions of the human gut and its associated organs, and may also facilitate differentiation-based strategies for treatment of a variety of disease states. [unreadable] [unreadable] [unreadable]